Regulator/promoter for tunable gene expression and metabolite sensing

ABSTRACT

A method for the facile and inexpensive inducible expression of heterologous genes has been discovered. The yhcS regulator gene has been found to be inducible by aromatic carboxylic acids and to alter the expression of operons in the LysR gene family, including the yhcRQP operon, common in enteric bacteria. Heterologous nucleic acid molecules placed under the control of yhcs responsive promoters may be overexpressed in response to the presence of inexpensive aromatic carboxylic acids.

[0001] This application claims the benefit of United States ProvisionalApplication No. 60/440,965 filed Jan. 17, 2003, which is herebyincorporated in its entirety by reference.

FIELD OF INVENTION

[0002] The present invention relates to the fields of molecular biologyand microbiology. More specifically, this invention pertains to a novelsystem for controlling gene expression levels that is induced byinexpensive, environmentally friendly, small molecules.

BACKGROUND

[0003] There is a need in the field of microbial metabolic engineeringfor tunable promoters and novel regulatory switches for the inducibleexpression of heterologous proteins. The “old standard” lac promoter/Lacrepressor system is still widely used. However, the commonly usedinducer, isopropylthio-beta-D-galactoside (“IPTG”), is expensive andthus not practical for large-scale bioprocesses. Additionally, higherconcentrations of IPTG increase the metabolic burden on the cell, inturn reducing the maximal expression of the target gene (Donovan et al.,J. Ind. Microbiol. 16:145-154 (1996)). The few available alternativesalso have limitations.

[0004] One possible solution to this problem is to appropriate existinggenetic systems of transcriptional regulators to enhance heterologousgene expression. A number a of transcriptional regulators are known. Forexample, the LysR family of transcriptional regulators is one of thelargest groups of transcriptional regulators in prokaryotes (Schell,Annu. Rev. Microbiol 47:597-626 (1993)). Currently, there are over 80known members of this regulator family. Proteins having greater than 20%amino acid identity with another LysR family member or having theconsensus sequence of the N-terminal region of the LysR family areconsidered to be members of this regulator family. LysR family membersare also commonly found in the size range of 276 to 324 amino acids,bind to similar DNA sequences in the absence of inducers, have promotersthat are located close to or overlapping those of the regulated targetgene, and most can repress their own transcriptional levels 3- to10-fold. Activation of the regulated target gene occurs in the presenceof inducer and usually results in a 6- to 200- fold increase inregulated target gene transcription. Regulated target genes are diverseand have numerous functions.

[0005] Recently, the gene encoded by open reading frame (“ORF”) b3243 inEscherichia coli (“E. coli”) has been demonstrated to function viaquorum sensing (Sperandio et al., Infect. Immun. 70:3085-3093 (2002)).Quorum sensing is the ability of bacteria cells to communicate with oneanother through perception of the accumulation of signaling moleculesbased on bacteria cell density. The gene encoded by ORF b3243 isup-regulated via quorum sensing resulting in a 23-fold increase intranscription of the gene. The protein produced by the b3243 ORF was notpurified. The gene was found to have a role in the regulation of the LEEgenes involved in a type III secretion system, a pathogenecity systemthat serves to translocate, upon contact with eukaryotic host cells,proteins from the bacteria cytoplasm into the host cell cytoplasm. Asthe b3243 ORF gene is a putative regulator of the LysR family, the b3243ORF gene itself was able to induce a four-fold induction of LEE1transcription. No inducer of the b3243 ORF gene was identified. One inthe art will appreciate that this inefficient induction prevents theb3243 ORF gene from being a viable promoter/regulator system without theidentification of its inducer.

[0006] The use of promoter/reporter gene constructs is well known in theart (Serebriiskii and Golemis, Anal. Biochem. 285:1-15 (2000); Spergelet al., Prog. Neurobiol. 63:673-686 (2001); Yarranton, Curr. Opin.Biotechnol. 3:506-511 (1992)). Particularly, reporter systems utilizingβ-galactosidase, green florescent protein, luciferase, andchloramphenicol acetyl transferase (CAT) are all commonly used in theart. Additionally, reporter systems such as luxCDABE are useful becausethis operon contains all of the genes required for bioluminescentreporting ((Van Dyk et al., Proc. Nat. Acad. Sci. USA 98:2555-2560(2001)).

[0007] The luxCDABE operon has been utilized to create a collection ofrandom gene fusions, comprising 27% of the known or predictedtranscriptional units of E. coli (Van Dyk et al., J. Bacteriol.183:5496-5505 (2001)). Treatment of E. coli cells containing these genefusions with nalidixic acid, a quinolone, results in selectiveup-regulation of ten genes. Some of these up-regulated genes areLexA-regulated SOS genes, while others are not generally induced by DNAdamage.

[0008] Aromatic compounds such as aromatic carboxylic acids are usuallytoxic to microorganisms. Numerous bacterial strains resistant toaromatic compounds, however, are known in the prior art (Diaz et al.,Microbiol. Mol. Biol. Rev. 65:523-569 (2001)). Additionally, a LysRfamily member from Acinetobacter, BenM, is responsive to synergisticinduction by benzoic acid, an aromatic carboxylic acid, and muconic acid(Bundy et al., Proc. Natl. Acad. Sci. USA 99:7693-7698 (2002)). Benzoicacid alone, however, produces minimal, if any, induction of BenMactivity. Even the synergistic response with muconic acid produces onlya four-fold increase in BenM activity.

[0009] U.S. Pat. No. 5,292,643 issued to Shibano et al. on Mar. 8, 1994describes genes related to fusaric acid resistance in variety ofmicroorganisms. Specifically, genes capable of decomposing ordetoxifying fusaric acid are disclosed. One of the genes postulated tobe involved in fusaric acid resistance, fusB, shares some homology withthe putativTe efflux transporter (PET) yhcP gene (Paulsen et al., FEMSMicrobiol. Lett. 156:1-8 (1997)). Applicants incorporate by referencethe co-owned and concurrently filed application entitled “PET Family ofEfflux Proteins”, U.S. patent application Ser. No. 60/440,760, whichdescribes new proteins efflux proteins whose expression may alter theexpression of carboxylic acids.

[0010] The problem to be solved therefore is to discover facile andinexpensive methods of inducible expression of heterologous genes.Applicants have solved the stated problem through the discovery that thepromoter elements of the yhcRQR operon are responsive to the expressionof the yhcS regulator (a member of the LysR family of transcriptionalregulators) whose expression may be induced by an inexpensive cadre ofaromatic carboxylic acid inducers.

SUMMARY OF THE INVENTION

[0011] The invention relates to the discovery that the yhcS regulator isresponsible for the activation of the yhcRQR operon promoter and isinducible by aromatic carboxylic acids, compounds that are typicallytoxic to cells. The expression of heterologous genes, placed under thecontrol of a yhcRQR operon promoter may be regulated by the presence ofan aromatic carboxylic acid. Accordingly the invention provides a methodfor the inducible expression of a heterologous nucleic acid moleculecomprising:

[0012] a) providing a host cell having a genome comprising:

[0013] i) a yhcS regulator gene responsive to an aromatic carboxylicacid inducer;

[0014] ii) a promoter region, responsive to expression of the yhcSregulator gene; and

[0015] iii) at least one heterologous nucleic acid molecule;

[0016] wherein the at least one heterologous nucleic acid molecule isoperably linked to the promoter region;

[0017] b) contacting the host cell of (a) with an aromatic carboxylicacid inducer wherein the at least one heterologous nucleic acid moleculeis expressed.

[0018] In a preferred embodiment the invention provides a method for theinducible expression of a heterologous nucleic acid molecule comprising:

[0019] a) providing an enteric bacterial host cell having a genomecomprising:

[0020] i) a yhcS regulator gene responsive to an aromatic carboxylicacid inducer;

[0021] ii) a promoter region, responsive to expression of the yhcSregulator gene; and

[0022] iii) at least one heterologous nucleic acid molecule;

[0023] wherein the at least one heterologous nucleic acid molecule isoperably linked to the promoter region;

[0024] b) contacting the host cell of (a) with an aromatic carboxylicacid inducer wherein the at least one heterologous nucleic acid moleculeis expressed.

[0025] Specific yhcS regulator gene useful in the present invention arethose that are selected from the group consisting of:

[0026] a) an isolated nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:1; and

[0027] b) an isolated nucleic acid molecule, which hybridizes to SEQ IDNO:1 after being washed with 0.1×SSC, 0.1% SDS at 65° C. and washed with2×SSC, 0.1% SDS followed by a second wash in 0.2×SSC, 0.1% SDS.

[0028] Similarly specific promoter responsive to expression of the yhcSregulator gene are those selected from the group consisting of:

[0029] a) an isolated nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:3; and

[0030] b) an isolated nucleic acid molecule, which hybridizes to SEQ IDNO:3 after being washed with 0.1×SSC, 0.1% SDS at 65° C. and washed with2×SSC, 0.1% SDS followed by a second wash in 0.2×SSC, 0.1% SDS.

[0031] In another embodiment the invention provides a host cellcomprising:

[0032] a) a yhcS regulator gene responsive to an aromatic carboxylicacid inducer having a nucleic acid sequence selected from the groupconsisting of:

[0033] i) an isolated nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:1; and

[0034] ii) an isolated nucleic acid molecule, which hybridizes to SEQ IDNO:1 after being washed with 0.1×SSC, 0.1% SDS at 65° C. and washed with2×SSC, 0.1% SDS followed by a second wash in 0.2×SSC, 0.1% SDS;

[0035] b) a promoter region, responsive to expression of the yhcSregulator gene having a nucleic acid sequence selected from the groupconsisting of:

[0036] i) an isolated nucleic acid molecule comprising nucleic acidsequence SEQ ID NO:3; and

[0037] ii) an isolated nucleic acid molecule, which hybridizes to SEQ IDNO:3 after being washed with 0.1 ×SSC, 0.1% SDS at 65° C. and washedwith 2×SSC, 0.1% SDS followed by a second wash in 0.2×SSC, 0.1% SDS; and

[0038] iii) at least one heterologous nucleic acid molecule;

[0039] wherein the at least one heterologous nucleic acid molecule isoperably linked to the promoter region.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

[0040] The invention can be more fully understood from the followingdetailed description, figures and the accompanying sequencedescriptions, which form a part of this application.

[0041]FIG. 1 shows the kinetics of the yhcRQP-luxCDABE response to PHBAin yhcS⁺and yhcS⁻host strains.

[0042] The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) andconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

[0043] SEQ ID NO:1 is the nucleotide sequence of the yhcS regulator.

[0044] SEQ ID NO:2 is the amino acid sequence of the YhcS protein.

[0045] SEQ ID NO:3 is the nucleotide sequence of the promoter regionupstream of yhcQ.

[0046] SEQ ID NO:4 is the nucleotide sequence of the primerKan-2FP(PCR).

[0047] SEQ ID NO:5 is the nucleotide sequence of the primerKan-2RP(PCR).

[0048] SEQ ID NO:6 is the nucleotide sequence of the primer Kan-2FP-1.

[0049] SEQ ID NO:7 is the nucleotide sequence of the primer Kan-2RP-1.

[0050] SEQ ID NO:8 is the nucleotide sequence of the primer YhcS.F.

[0051] SEQ ID NO:9 is the nucleotide sequence of the primer YhcS.R.

DETAILED DESCRIPTION OF THE INVENTION

[0052] There is a need in the field of microbial metabolic engineeringfor tunable promoters and novel regulatory switches. Advantages ofApplicants' system are the very low basal levels of gene expression inthe absence of inducer and expression levels that vary with theconcentration of inducer up to relatively high expression levels.Furthermore, the cost of many of the inducing molecules is relativelyinexpensive.

[0053] There are numerous uses for Applicants' tunable promoter systemcomprising the YhcS regulatory protein and the responsive promoterregion. This novel promoter/regulator system can be used to control andregulate expression of genes and operons of interest by applyingstandard molecular biology methods as has been demonstrated herein bythe controlled expression of luxCDABE.

[0054] Another possible application is suggested by analogy to otherLysR family proteins, which typically have a conformational change uponbinding their cognate inducer. YhcS protein likely binds to certainaromatic carboxylic acid molecules and changes conformation such that itactivates gene expression upon DNA binding. This conformation change maybe useful to sense small molecules in applications such as those foundin nano scale systems.

[0055] The range of molecules to which YhcS responds may be manipulatedby application of a variety of protein engineering techniques. Thus, theuseful range of molecules of each of the above-mentioned applicationscould be expanded.

[0056] Advantages of this regulator/promoter system include, but are notlimited to, basal expression levels that are extremely low; high levelsof expression following induction; inexpensive, environmentally friendlyinducers; when used in conjunction with other expression systems, analternative expression system that will allow differential regulation ofvarious genes in a genetically engineered host; and a proteinconformation change useful for nanotechnology.

[0057] Applicants specifically incorporate the entire content of allcited references in this disclosure.

[0058] In the context of this disclosure, a number of terms shall beutilized.

[0059] The term “pHBA” is the abbreviation for para-hydroxybenzoic acid,which is also known as para-hydroxybenzoate.

[0060] The term “PHCA” is the abbreviation for para-hydroxycinnamicacid, which is also known as para-hydroxycinnamate.

[0061] The term “CA” is the abbreviation for cinnamic acid, which isalso known as cinnamate

[0062] An “isolated nucleic acid molecule” refers to a polymer of RNA orDNA that is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. An isolated nucleic acidmolecule in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

[0063] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), particularly Chapter11 and Table 11.1 therein (entirely incorporated herein by reference)(hereinafter “Sambrook”). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Stringencyconditions can be adjusted to screen for moderately similar fragments,such as homologous sequences from distantly related organisms, to highlysimilar fragments, such as genes that duplicate functional enzymes fromclosely related organisms. Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS is increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C and washedwith 2×SSC, 0.1% SDS followed by a second wash in 0.2×SSC, 0.1% SDS, forexample.

[0064] Hybridization requires that the two nucleic acids containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of Tm for hybrids of nucleicacids having those sequences. The relative stability (corresponding tohigher Tm) of nucleic acid hybridizations decreases in the followingorder: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100nucleotides in length, equations for calculating Tm have been derived(Sambrook supra). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (Sambrooksupra). In one embodiment the length for a hybridizable nucleic acid isat least about 10 nucleotides. Preferably, a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

[0065] A “substantial portion” refers to an amino acid or nucleotidesequence which comprises enough of the amino acid sequence of apolypeptide or the nucleotide sequence of a gene to afford putativeidentification of that polypeptide or gene, either by manual evaluationof the sequence by one skilled in the art, or by computer-automatedsequence comparison and identification using algorithms such as BLAST(Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol.215:403-410 (1993). In general, a sequence of ten or more contiguousamino acids or thirty or more nucleotides is necessary in order toputatively identify a polypeptide or nucleic acid sequence as homologousto a known protein or gene. Moreover, with respect to nucleotidesequences, gene specific oligonucleotide probes comprising 20-30contiguous nucleotides may be used in sequence-dependent methods of geneidentification (e.g., Southern hybridization) and isolation (e.g., insitu hybridization of bacterial colonies or bacteriophage plaques). Inaddition, short oligonucleotides of 12-15 bases may be used asamplification primers in PCR in order to obtain a particular nucleicacid molecule comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence toafford specific identification and/or isolation of a nucleic acidmolecule comprising the sequence. The instant specification teachespartial or complete amino acid and nucleotide sequences encoding one ormore particular bacterial proteins. The skilled artisan, having thebenefit of the sequences as reported herein, may now use all or asubstantial portion of the disclosed sequences for the purpose known tothose skilled in the art. Accordingly; the instant invention comprisesthe complete sequences as reported in the accompanying Sequence Listing,as well as substantial portions of those sequences as defined above.

[0066] The term “complementary” describes the relationship betweennucleotide bases that are capable to hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid molecules that are complementary tothe complete sequences as reported in the accompanying Sequence Listingas well as those substantially similar nucleic acid sequences.

[0067] The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press, New York (1987); and Sequence Analysis Primer(Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG Pileupprogram found in the GCG program package, using the Needleman and Wunschalgorithm with their standard default values of gap creation penalty=12and gap extension penalty=4 (Devereux et al., Nucleic Acids Res.12:387-395 (1984)), BLASTP, BLASTN, and FASTA (Pearson et al, Proc.Natl. Acad. Sci. USA 85:2444-2448 (1988). The BLASTX program is publiclyavailable from NCBI and other sources (BLAST Manual, Altschul et al.,Natl. Cent. Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH,Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990);Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). Anotherpreferred method to determine percent identity is by the method ofDNASTAR protein alignment protocol using the Jotun-Hein algorithm (Heinet al., Meth. Enzymol. 183:626-645 (1990)). Default parameters for theJotun-Hein method for alignments are: for multiple alignments, gappenalty=11, gap length penalty=3; for pairwise alignments ktuple=6. Asan illustration, by a polynucleotide having a nucleotide sequence havingat least, for example, 95% “identity” to a reference nucleotide sequenceit is intended that the nucleotide sequence of the polynucleotide isidentical to the reference sequence except that the polynucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with another nucleotideor a number of nucleotides up to 5% of the total nucleotides in thereference sequence may be inserted into the reference sequence. Thesemutations of the reference sequence may occur at the 5′ or 3′ terminalpositions of the reference nucleotide sequence or anywhere between thoseterminal positions, interspersed either individually among nucleotidesin the reference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid. In other words, toobtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy-terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

[0068] The term “percent homology” refers to the extent of amino acidsequence identity between polypeptides. When a first amino acid sequenceis identical to a second amino acid sequence, then the first and secondamino acid sequences exhibit 100% homology. The homology between any twopolypeptides is a direct function of the total number of matching aminoacids at a given position in either sequence, e.g., if half of the totalnumber of amino acids in either of the two sequences is the same thenthe two sequences are said to exhibit 50% homology. “Synthetic genes”can be assembled from oligonucleotide building blocks that arechemically synthesized using procedures known to those skilled in theart. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

[0069] “Gene” refers to a nucleic acid molecule that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature.

[0070] “Genome” refers to the entire genetic information containedwithin an organism (e.g., chromosome, plasmid, plastid, or mitochondrialDNA). “Endogenous gene” refers to a native gene in its natural locationin the genome of an organism. A “foreign” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure. “Structural gene” refers to a gene that codes for the aminoacid sequence of a protein or for a ribosomal RNA or transfer RNA. An“operon” refers to a controllable unit of transcription consisting of anumber of structural genes transcribed together.

[0071] “Coding sequence” refers to a DNA sequence that codes for aspecific amino acid sequence. “Suitable regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0072] “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

[0073] “Heterologous” as used in the context of gene expression relatesto that which is “foreign” to a particular environment. Thus, a“heterologous gene” or “heterologous nucleic acid molecule” means anucleic acid molecule that is foreign, or non-native to a particularhost or genome. A “heterologous protein” is a protein that is foreign toa host cell and is typically encoded by a heterologous gene.Heterologous nucleic acids of the invention are typically expressedunder the control of regulated promoters in an inducible fashion.

[0074] The term “regulator” refers to a protein whose primary functionis to control the rate of expression of regulated genes. Regulation ofgene expression can be by positive activation or by repression. A“regulatory gene” refers to a gene that encodes a regulator. Within thecontext of te present invention a typical regulator gene is the yhcSgene

[0075] “Host cell” refers to a cell into which has been introduced(e.g., transformed or transfected) an exogenous polynucleotide sequence,i.e. a heterologogus nucleic acid molecule. Host cells are typicallyprokaryotic cells such as bacteria, e.g., E. coli, and may be eukaryoticcells such as yeast, insect, amphibian, green plant, or mammalian cells,where the relevant regulator genes exist.

[0076] “Inducer” refers to a small molecule that initiatestranscription, or increases the rate of transcription, of a desiredgene. Within the context of the present invention typical inducers arearomatic carboxylic acids having the ability to activate a regulatorgene.

[0077] “Reporter gene” refers to a gene that encodes an easily assayedproduct (e.g. luxCDABE, bgaB, cat, dsRed, galK, gfp, lacZ, luc, luxAB,nptll, phoA, uidA, or xylE). Typically reporters are coupled to thepromoter and/or regulator sequence of another gene and transfected intoa host cell. The reporter gene can then be used to see which factorsactivate response elements in the upstream region of the gene ofinterest.

[0078] “Translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner et al., Mol. Biotechnol. 3:225(1995)).

[0079] “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by lngelbrecht et al., Plant Cell1:671-680 (1989).

[0080] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. “Functional RNA”refers to antisense RNA, ribozyme RNA, or other RNA that is nottranslated yet and has an effect on cellular processes.

[0081] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid molecule so that the function ofone is affected by the other. For example, a promoter is operably linkedwith a coding sequence when it affects the expression of that codingsequence (i.e., that the coding sequence is under the transcriptionalcontrol of the promoter). Coding sequences can be operably linked toregulatory sequences in sense or antisense orientation.

[0082] The term “expression” refers to the transcription and stableaccumulation of sense (mRNA) or antisense RNA derived from the nucleicacid molecule of the invention. Expression may also refer to translationof mRNA into a polypeptide. “Antisense inhibition” refers to theproduction of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or nontransformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020).

[0083] “Transformation” refers to the transfer of a nucleic acidmolecule into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms.

[0084] The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

[0085] “PCR” or “polymerase chain reaction” is a technique used for theamplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and4,800,159).

[0086] Standard recombinant DNA and molecular cloning techniques usedhere are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) (hereinafter “Sambrook”); and by Silhavy, T. J., Bennan, M.L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-lnterscience (1987).

[0087] The present invention relates to the discovery that aromaticcarboxylic acids induce the up-regulation or expression of the regulatorgene yhcS which in turn is responsible for the activation of thepromoter region driving expression of the yhcRQP operon. Expression ofheterologous nucleic acid molecules which are operably to thesepromoters may therefore be inducibly regulated by the presence orabsence of inexpensive aromatic carboxylic acids in the medium.

[0088] Regulator Systems

[0089] The yhcS regulator and the corresponding yhcRQR operon is knownand is common in a variety of enteric bacteria such as Escherichia(Hayashi et al., “Complete genome sequence of enterohemorrhagicEscherichia coli O157:H7 and genomic comparison with a laboratory strainK-12”, DNA Res. 8 (1), 11-22 (2001)); Yersinia (Parkhill et al., “Genomesequence of Yersinia pestis, the causative agent of plague”, Nature 413(6855), 523-527 (2001)); Shigella (Wei et al., “Complete Genome Sequenceand Comparative Genomics of Shigella flexneri Serotype 2a Strain 2457T”,Infect. Immun. 71 (5), 2775-2786 (2003)); and Salmonella (McClelland etal., “Complete genome sequence of Salmonella enterica serovarTyphimurium LT2”; Nature 413 (6858), 852-856 (2001)). It will beappreciated by one of skill in the art that those organisms havinghomologs to the present regulators will be expected to function inheterologous gene expression in similar ways.

[0090] Those cells having existing homologous regulator systems may beused in the present invention for the expression of heterologous DNAsimply by the insertion of the DNA to be expressed in the appropriateposition in the genome and in the correct orientation for expression.Thus a Salmonella or Shigella strain, as described above, may be used inthis fashion. Host cells suitable for use in the present invention willinclude but are not limited to Escherichia, Salmonella, Bacillus,Acinetobacter, Streptomyces, Methylobacter, Rhodococcus,Corynebacterium,Pseudomonas, Rhodobacter, and Synechocystis.

[0091] Particularly suitable in the present invention are members of theenteric class of bacteria. Enteric bacteria are members of the familyEnterobacteriaceae and include such members as Escherichia, Salmonella,and Shigella. They are gram-negative straight rods, 0.3-1.0×1.0-6.0 mm,motile by peritrichous flagella (except for Tatumella) or nonmotile.They grow in the presence and absence of oxygen and grow well onpeptone, meat extract, and (usually) MacConkey's media. Some grow onD-glucose as the sole source of carbon, whereas others require vitaminsand/or mineral(s). They are chemoorganotrophic with respiratory andfermentative metabolism but are not halophilic. Acid and often visiblegas is produced during fermentation of D-glucose, other carbohydrates,and polyhydroxyl alcohols. They are oxidase negative and, with theexception of Shigella dysenteriae 0 group 1 and Xenorhabdusnematophilus, catalase positive. Nitrate is reduced to nitrite (exceptby some strains of Erwinia and Yersina). The G+C content of DNA is 38-60mol % (T_(m), Bd). DNAs from species within most genera are at least 20%related to one another and to Escherichia coli, the type species of thefamily. Notable exceptions are species of Yersina, Proteus, Providenica,Hafnia and Edwardsiella, whose DNAs are 10-20% related to those ofspecies from other genera. Except for Erwinia chrysanthemi, all speciestested contain the enterobacterial common antigen (Bergy's Manual ofSystematic Bacteriology, D. H. Bergy et al., Baltimore: Williams andWilkins, 1984).

[0092] It is clear that host cells comprising the present regulatorsystems are suitable for use in the invention. However, where it isdesired to find new strains having the present regulator systems, or toidentify new regulator genes having greater functionality in non-nativehost cells, it will be possible to use the sequence information providedin the literature and in this disclosure to identify and isolate suchhomologs.

[0093] Isolation of Homologs

[0094] A specific yhcS regulator has been identified in the E. coligenome and has the nucleic acid sequence as set forth in SEQ ID NO:1.The promoter region of the yhcRQP operon, responsive to the expressionof this yhcS regulator has the nucleic acid sequence as set forth in SEQID NO:3. It will be apparent to the skilled artisan that homologs to theE. coli sequences or others cited in the literature may easily beidentified based on current practices in molecular biology, and suchhomologs will be equally applicable and useful in the present invention.For example, one of skill in the art may use the nucleic acid moleculesof the instant invention to isolate cDNAs and genes encoding ahomologous YhcS protein from the same or other bacterium species.Isolation of homologous genes using sequence-dependent protocols is wellknown in the art.

[0095] Examples of sequence-dependent protocols include, but are notlimited to, methods of nucleic acid hybridization, and methods of DNAand RNA amplification as exemplified by various uses of nucleic acidamplification technologies (e.g., PCR or ligase chain reaction).

[0096] For example, yhcS gene, either as cDNA or genomic DNA, could beisolated directly by using all or a portion of the instant nucleic acidmolecule as DNA hybridization probes to screen libraries from anydesired bacterium employing methodology well known to those skilled inthe art. Specific oligonucleotide probes based upon the instant yhcSgene sequence can be designed and synthesized by methods known in theart (Sambrook supra). Moreover, the entire sequences can be useddirectly to synthesize DNA probes by methods known to the skilledartisan such as random primers DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part of or full- length of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

[0097] In addition, two short segments of the instant nucleic acidmolecule may be used in polymerase chain reaction protocols to amplifylonger nucleic acid molecules encoding homologous yhcS genes from DNA orRNA. The polymerase chain reaction may also be performed on a library ofcloned nucleic acid molecules wherein the sequence of one primer isderived from the instant nucleic acid molecules, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor. Alternatively, the secondprimer sequence may be based upon sequences derived from the cloningvector. For example, the skilled artisan can follow the RACE protocol(Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998-9002 (1988)) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (Invitrogen,Carlsbad, Calif.), specific 3′ or 5′ cDNA fragments can be isolated(Ohara et al., Proc. Natl. Acad. Sci. USA 86:5673-5677 (1989); Loh etal., Science 243:217-220 (1989)). Products generated by the 3′ and 5′RACE procedures can be combined to generate full-length cDNAs (Frohmanet al., Techniques 1:165 (1989)).

[0098] Alternatively the yhcS sequences may be employed as anhybridization reagent for the identification of homologs. The basiccomponents of a nucleic acid hybridization test include a probe, asample suspected of containing the gene or gene fragment of interest,and a specific hybridization method. Probes are typically singlestranded nucleic acid sequences which are complementary to the nucleicacid sequences to be detected. Probes are “hybridizable” to the nucleicacid sequence to be detected. The probe length can vary from 5 bases totens of thousands of bases, and will depend upon the specific test to bedone. Typically a probe length of about 15 bases to about 30 bases issuitable. Only part of the probe molecule need be complementary to thenucleic acid sequence to be detected. In addition, the complementaritybetween the probe and the target sequence need not be perfect.Hybridization does occur between imperfectly complementary moleculeswith the result that a certain fraction of the bases in the hybridizedregion are not paired with the proper complementary base.

[0099] Hybridization methods are well defined. Typically the probe andsample must be mixed under conditions which will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration theshorter the hybridization incubation time needed. Optionally achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness and Chen, Nucl. Acids Res.19:5143-5151(1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide, and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

[0100] Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1 Mbuffers, such as sodium citrate, Tris-HCI, PIPES or HEPES (pH rangeabout 6-9), about 0.05 to 0.2% detergent, such as sodium dodecylsulfate,or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500kilodaltons), polyvinylpyrrolidone (about 250-500 kdal), and serumalbumin. Also included in the typical hybridization solution will beunlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmentednucleic DNA, e.g., calf thymus or salmon sperm DNA, or yeast RNA, andoptionally from about 0.5 to 2% wt./vol. glycine. Other additives mayalso be included, such as volume exclusion agents which include avariety of polar water-soluble or swellable agents, such as polyethyleneglycol, anionic polymers such as polyacrylate or polymethylacrylate, andanionic saccharidic polymers, such as dextran sulfate.

[0101] Nucleic acid hybridization is adaptable to a variety of assayformats. One of the most suitable is the sandwich assay format. Thesandwich assay is particularly adaptable to hybridization undernon-denaturing conditions. A primary component of a sandwich-type assayis a solid support. The solid support has adsorbed to it or covalentlycoupled to it immobilized nucleic acid probe that is unlabeled andcomplementary to one portion of the sequence.

[0102] Heterologous DNA Expression

[0103] Once a host cell comprising a yhcS regulator and the responsiveyhcRQP operon has been identified or constructed it will be necessary toinsert the heterologous nucleic acid molecule into the genome in theposition that will be appropriate for expression. Methods for thetransformation of microbial cells and integration of DNA into a genomeare common and well known in the art (see Sambrook supra). Typically atransformation vector is constructed for this purpose containing theessential elements for transformation and DNA integration. Typically thevector or cassette contains sequences directing transcription andtranslation of the relevant gene, a selectable marker, and sequencesallowing autonomous replication or chromosomal integration. Suitablevectors comprise a region 5′ of the gene which harbors transcriptionalinitiation controls and a region 3′ of the DNA fragment which controlstranscriptional termination. It is most preferred when both controlregions are derived from genes homologous to the transformed host cell,although it is to be understood that such control regions need not bederived from the genes native to the specific species chosen as aproduction host.

[0104] Heterologous nucleic acid molecules suitable for expression bythe methods of the invention are virtually unlimited. In some instancesthe heterologous nucleic acid molecule may not encode a protein and beexpressed for the purpose of controlling or suppressing (as in antisenseorientation for example) other genetic elements in the genome. Morecommonly, the foreign DNA will encode a protein and typically an enzymethat is part of a pathway. It will be appreciated that a single DNAfragment may be expressed by the present method or several linkedfragments comprising all or a part of an enzymatic pathway. It istherefore within the scope of the present invention to expressheterologous nucleic acid molecules encoding at least one proteinwherein the at least one protein is part of an enzymatic biosyntheticpathway producing a product selected from the group consisting ofisoprenoids; terpenoids, tetrapyrroles, polyketides, vitamins, aminoacids, fatty acids, proteins, nucleic acids, carbohydrates,antimicrobial agents, and anticancer agents.

[0105] In some instances it will be useful to monitor the expression oractivation of the regulator systems through the use of a reporter.Reporters are common and well known in the art and a non-inclusive listof those suitable in the present invention are luxCDABE, bgaB, cat,dsRed, galK, gfp, lacZ, luc, luxAB, nptll, phoA, uidA, and xylE.

[0106] One of the key advantages of the present invention is the abilityto control the expression of the heterologous DNA by the action of aninducer. Applicant's discovery that the present regulator genes areresponsive to aromatic carboxylic acids is fortuitous in that thesecompounds are inexpensive and easily obtained. Any aromatic carboxylicacid will have utility in the present method where para-hydroxybenzoicacid, para-hydroxycinnamic acid, cinnamic acid, salicylic acid, benzoicacid, and 1-napthoic acid are preferred.

[0107] Description of the Preferred Embodiments

[0108] As described in the following examples, during the course ofLuxArray and DNA array analyses, Applicants discovered that expressionof an E. coli operon, yhcRQP, was highly induced by treatment of E. colicells with aromatic carboxylic acids, such a para-hydroxybenzoic acid(pHBA), para-hydroxycinnamic acid (pHCA), and cinnamic acid (CA). AluxCDABE gene fusion with the promoter region of this operon controllingthe bioluminescent reporter was used to characterize expression of thisoperon. In the absence of inducing molecules the expression level wasvery low, just slightly above background. However, when the cell wastreated with inducer, the expression level was dramatically elevated,where expression increased nearly 1000-fold. This represents much higherexpression than found in typical LysR family members. Furthermore, theexpression level was dependent on the concentration of inducer added.

[0109] The gene immediately upstream of the yhcRQP operon at openreading frame b3243, YhcS, is a putative member of the LysR family oftranscription regulators. Using a transposon insertion mutation in yhcS,expression of the yhcRQP-luxCDABE gene fusion was no longer induced byaromatic carboxylic acids. Thus, it was shown that yhcS encodes apositive-acting transcriptional regulator responsible for the dramatic,tunable changes in gene expression of the yhcRQP operon.

[0110] This promoter/regulator system can be used to control andregulate expression of other genes and operons of interest by applyingstandard molecular biology methods. Furthermore, by analogy to otherLysR family proteins, the YhcS protein likely binds to these aromaticcarboxylic acid molecules and changes conformation such that itactivates gene expression upon DNA binding. This conformation change maybe useful to sense small molecules in nano-scale systems.

EXAMPLES

[0111] The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesin Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usage and conditions.

[0112] General Methods

[0113] Standard recombinant DNA and molecular cloning techniques used inthe Examples are well known in the art and are described by Sambrook,J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.(1989); by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experimentswith Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols inMolecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience, Hoboken, N.J. (1987).

[0114] Standard genetic methods for transduction used in the Examplesare well known in the art and are described by Miller, J. H.,Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1972).

[0115] The meaning of abbreviations is as follows: “kb” meanskilobase(s), “hr” means hour(s), “min” means minute(s), “sec” meanssecond(s), “d” means day(s), “ml” means milliliter(s), “μl” meansmicroliter(s), “nl” means nanoliter(s), “μg” means microgram(s), “ng”means nanogram(s), “mM” means millimolar, “μM” means micromolar, “nm”means nanometer(s), “OD₆₀₀” means the optical density measured at awavelength of 600 nm, “RLU” means relative light units.

[0116] Media and Culture Conditions:

[0117] Materials and methods suitable for the maintenance and growth ofbacterial cultures were found in Experiments in Molecular Genetics(Jeffrey H. Miller), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1972); Manual of Methods for General Bacteriology (PhillipGerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, WillisA. Wood, Noel R. Krieg and G. Briggs Phillips, eds), pp. 210-213,American Society for Microbiology, Washington, D.C. (1981); or Thomas D.Brock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass. All reagentsand materials used for the growth and maintenance of bacterial cellswere obtained from Aldrich Chemicals (Milwaukee, Wis.), BD DiagnosticSystems (Sparks, Md.), Invitrogen Corp. (Carlsbad, Calif.), or SigmaChemical Company (St. Louis, Mo.) unless otherwise specified.

[0118] LB medium contains the following per liter of medium:Bacto-tryptone (10 g), Bacto-yeast extract (5 g), and NaCl (10 g).

[0119] Vogel-Bonner medium contains the following per liter: 0.2 gMgSO₄·7H₂O, 2 g citric acid·1H₂O, 10 g K₂HPO₄ and 3.5 g NaNH₄HPO₄·4H₂O.

[0120] Minimal M9 medium contains the following per liter of medium:Na₂HPO₄ (6 g), KH₂PO₄ (3 g), NaCl (0.5 g), and NH₄Cl (1 g).

[0121] Above media were autoclaved for sterilization then 10 ml of 0.01M CaCl₂ and 1 ml of 1 M MgSO₄·7H₂O were added to M9 medium. Vitamin B1(thiamin) was added at 0.0001% to both Vogel-Bonner and M9 media. Carbonsource and other nutrients and supplements were added as mentioned inthe Examples. All additions were pre-sterilized before they were addedto the media.

[0122] Molecular Biology Techniques:

[0123] Restriction enzyme digestions, ligations, transformations, andmethods for agarose gel electrophoresis were performed as described inSambrook supra. Polymerase Chain Reactions (PCR) techniques were foundin White, B., PCR Protocols: Current Methods and Applications, Volume 15(1993) Humana Press Inc, Totowa, N.J.

Example 1 Gene Expression Profiling of para-hydroxybenzoate-treated E.coli Cells

[0124] The alterations in the E. coli gene expression profile uponexposure to pHBA were examined using DNA microarray technology. E. colistrain DE112 (Van Dyk et al. Appl. Environ. Microbiol. 60:1414-1420(1994)) was grown in Vogel-Bonner minimal medium with glucose as acarbon source to an OD₆₀₀ of 0.2. At this point the culture was split intwo flasks and PHBA in the acid form was added to one flask from a stocksolution in ethanol to achieve a final PHBA concentration of 25 mM. ThepH of the medium in the flask with PHBA was lowered by an unmeasuredamount. An equivalent volume of ethanol without PHBA was added to theother flask. Approximately 58% growth inhibition resulted from the PHBAtreatment under these conditions. Cells were harvested from the controland treated flasks at 30 and 60 minutes after pHBA addition. RNAisolation, array hybridization, and data analysis were done aspreviously described (Wei et al, J. Bacteriol. 183:545-556 (2001),Smulski et al. J. Bacteriol. 183:3353-3364 (2001)). Among the genes thatwere highly induced by this treatment at the 60 minute time point werethe yhcR, yhcQ, and yhcP genes (Table 1). The experiment was repeated,and again, these three genes were highly upregulated (Table 2). Thesethree genes are predicted to be transcribed as an operon. Thereproducible observed co-regulation in response to PHBA treatment isconsistent with this prediction. Thus, these three genes will bereferred to as the yhcRQP operon. TABLE 1 Upregulation of genes in theyhcRQP operon after 60 minutes pHBA treatment, experiment 1 BlattnerSignal in Signal in Ratio Gene No.* untreated pHBA treated(treated/untreated) yhcR b3242 0.0190 1.37 72.3 yhcQ b3241 0.393 1.614.09 yhcP b3240 1.17 7.52 6.45

[0125] TABLE 2 Upregulation of genes in the yhcRQP operon after 60minutes pHBA treatment, experiment 2 Blattner Signal in Signal in pHBARatio Gene No.* untreated treated (treated/untreated) yhcR b3242 0.6877.18 10.5 yhcQ b3241 0.270 6.02 22.3 yhcP b3240 0.994 12.0 12.1

Example 2 LuxArray Analysis of Para-hydroxycinnamic Acid and CinnamicAcid Treated E. coli

[0126] Gene expression profiles were done using LuxArray version 1.04,which has been fully described (Van Dyk et al. J. Bacteriol.183:5496-5505 (2001), Gonye et al. U.S. patent application Publication20030219736). This method utilizes a set of bioluminescent gene fusionsto ⅓ of E. coli transcriptional units in a toIl host strain that ishypersensitive to many compounds, including pHCA. Sublethalconcentrations of PHCA, 10 mM and 5 mM, and CA, 8 mM and 4 mM, at whichstress and other responses can be detected using luxCDABE reporter genefusions, were used. Each of these treatments yielded nearly identicalexpression patterns, suggesting a similar cellular response to these twoaromatic compounds. A predominant feature observed in both theseprofiles was that one gene fusion, lux-a.pk035.c7, was the severalhundred-fold upregulated. The lux-a.pk035.c7 gene fusion contains an E.coli chromosomal segment between nucleotides 3385829 and 3386761according to the E. coli genomic sequence, which contains the promoterregion of the putative yhcRQP operon and the entire yhcR gene and the 5′end of the yhcQ gene. This chromosomal segment is joined to the luxCDABEgene fusion in the parental plasmid, pDEW201 (Gonye et al. U.S. patentapplication Publication 20030219736) thus forming junction between yhcQgene and luxC. Accordingly, this gene fusion will report on expressionof the yhcQ gene and any other genes cotranscribed with it. As detailedin Example 1, it is likely that yhcR, yhcQ, and yhcP form an operon,thus this gene fusion is referred to as a yhcRQP-luxCDABE gene fusion.The strain containing this gene fusion was given the name DPD2411, andthe plasmid within this strain that contains yhcRQP-luxCDABE gene fusionwas called pDEW655.

[0127] Table 3 shows the bioluminescent response of this reporter genefusion to pHCA, CA, and ethanol. As part of a larger LuxArrayexperiment, two independent, actively growing, cultures carrying theyhcRQP-luxCDABE gene fusion in LB medium were each split three ways attime zero. Two aliquots were treated with different concentrations ofeach chemical and the third aliquot was the untreated control. Thenormalized bioluminescent signal from each of two replicas in theLuxArray is shown for the measurements at four time points (in minutes).

[0128] The dramatic upregulation of expression in response to pHCA andCA treatments at each of the time points other than the initial, zerotime point is clear. In contrast, ethanol treatment does not induceincreased bioluminescence. TABLE 3 Responses of the yhcRQP-luxCDABE genefusion to pHCA, CA, or ethanol Replica 1 Replica 2 normalized RLUnormalized RLU Treatment 0 min 45 min 90 min 135 min 0 min 45 min 90 min135 min  0 mM pHCA 0.061 0.071 0.067 0.039 0.043 0.04 0.039 0.022  5 mMpHCA 0.056 10.695 14.276 13.533 0.043 9.325 14.754 10.587 10 mM pHCA0.056 9.094 14.314 15.283 0.034 7.31 11.164 12.867  0 mM CA 0.03 0.0340.024 0.013 0.08 0.052 0.032 0.015  4 mM CA 0.026 4.092 5.845 5.3940.062 2.928 4.951 3.678  8 mM CA 0.022 4.937 8.379 6.981 0.054 3.4056.193 5.746 0% ethanol 0.021 0.023 0.017 0.009 0.045 0.028 0.018 0.0083% ethanol 0.024 0.024 0.022 0.012 0.038 0.029 0.026 0.014 5% ethanol0.022 0.014 0.022 0.015 0.037 0.017 0.017 0.014

Example 3 Regulation of vhcRQP expression by YhcS

[0129] The yhcS gene of E. coli encodes an uncharacterized member of theLysR family of positive acting regulatory molecules. This gene islocated immediately adjacent to the yhcRQP operon that was found to beupregulated by PHBA treatment in DNA array experiments and by PHCA andCA treatments in LuxArray experiments. The possibility that YhcScontrols expression of yhcRQP was tested using a yhcS null mutation.

[0130] Such a mutation was found in an E. coli library of transposoninsertion mutations constructed using the transposome system based onthe Tn5 transposon (Epicentre, Madison, Wis.). A transposome is aprotein-DNA complex composed of the EZ::TN<Kan-1> transposon and theEZ::TN transposase. The EZ::TN transposase is bound to the ends of thetransposon, which facilitates the formation of a stable synapticcomplex. The transposome requires Mg⁺² to initiate the insertion of theEZ::TN<Kan-1> transposon into target DNA. The cellular levels of Mg⁺²are sufficient to activate the transposome. Thus, the electroporation ofthe transposome into cells permits the in vivo insertion of theEZ::TN<Kan-1> transposon into bacterial genomes.

[0131] The EZ::TN<Kan-1> transposome was electroporated intoelectroporation competent E. coli strain DH5αE cells (Invitrogen,Carlsbad, Calif.). Following electroporation, the cells were grown inSOC medium (Initrogen) for one hour at 37° C. with aeration.Subsequently, the cells were plated onto LB agar plates containingkanamycin (50 μg/ml) (LB+Kan) and incubated overnight at 37° C.Individual colonies were inoculated into 96-well microtiter platescontaining 150 μl of LB+Kan and incubated overnight at 37° C.

[0132] “Single Primer PCR” was used to determine the identity of each E.coli transposon mutation. Using a single DNA primer that wascomplementary to one end of the EZ::TN<Kan-1> transposon, PCR productswere generated. Subsequently, a second DNA primer (located internal andadjacent to the PCR primer) was used to sequence the PCR products. TheDNA primer used in the PCR reaction was either Kan-2FP(PCR) (SEQ IDNO:4) or Kan-2RP(PCR) (SEQ ID NO:5) and the DNA primer used for DNAsequencing was either Kan-2FP(PCR) (SEQ ID NO:4) or Kan-2RP(PCR) (SEQ IDNO:5), respectively. The PCR reaction conditions were the following: (1)94° C., 15 minutes (2) 20 cycles—94° C., 30 seconds; 60° C., 30 seconds;72° C., 3 minutes (3) 30 cycles—94° C., 30 seconds; 40° C., 30 seconds;72° C., 2 minutes (4) 30 cycles—94° C., 30 seconds; 60° C., 30 seconds;72° C., 2 minutes (5) 72° C., 7 minutes. The PCR reactions were preparedfor DNA sequencing using the QlAquick PCR Purification Kit (Qiagen,Valencia, Calif.).

[0133] The yhcS transposon mutant was identified using PCR amplificationprimer Kan-2FP(PCR) (SEQ ID NO:4) and DNA sequencing primer Kan-2FP-1(SEQ ID NO:6). The transposon mutation was confirmed using gene-specificprimers: YhcS.F (SEQ ID NO:8) and YhcS.R (SEQ ID NO:9) andtransposon-specific primers Kan-2FP-1 (SEQ ID NO:6) and Kan-2RP-1 (SEQID NO:7).

[0134] The size of the yhcS gene is ˜929 base pairs. The transposoninsertion site within the yhcS gene is ˜330 base pairs away from the 5′end of yhcS. A PCR reaction done with the YhcS.F and Kan-2RP-1 primersyielded a PCR fragment ˜550 base pairs and PCR primers YhcS.R andKan-2FP-1 yielded a PCR product <400 base pairs in size.

[0135]E. coli strain DPD2410 is the DH5αE derived strain containing theyhcS::TN<Kan> mutation. Strains DH5αE and DPD2410 were transformed withpDEW655 to generate E. coli strains DPD2413 and DPD2415, respectively. Asingle colony of each of these two strains from an LB plate containing150 μg/ml Ampicillin was used to inoculate 200 μl LB medium in wells ofa 96 well, white microplate (Microlite, Dynex Technologies, Chantilly,Va.). The plate was incubated for 90 minutes at 37° C.; then 50 μl ofthe cultures was added to 50 μl of LB medium or to 50 μl of LB mediumcontaining pHBA in the acid form, which had been added from a stocksolution in ethanol. The final concentration of pHBA was 5 mM and thefinal concentration of ethanol was 0.25%. The pH of the medium with PHBAwas lowered by an unmeasured amount. The bioluminescence was quantitatedwith a Luminoskan Ascent microplate luminometer (Thermo Labsystems,Franklin, Md.). The results of this study are presented in FIG. 1, whichis a plot of the bioluminescence intensity in relative light units (RLU)versus time in minutes. In the Figure, addition of PHBA was made at timezero. Solid lines are the response in the yhcS⁺ strain, DPD2413. Dottedlines are the response in the yhcS⁻ strain, DPD2415. Circles representPHBA treated cultures and triangles represent untreated cultures. FIG. 1clearly shows that pHBA induced rapid and dramatic upregulation of theyhcRQP-luxCDABE expression in the yhcS⁺ host strain, but that thisupregulation was essentially abolished in the yhcS::TN<Kan> host strain.

[0136] A derivative of E. coli strain MG1655 (obtained from Prof.Douglas Berg, Washington University School of Medicine, St. Louis, Mo.)with the yhcS::TN<Kan> mutation was made by P1clr100Cm mediatedtransduction using phage grown on strain DPD2410 as a donor andselection for kanamycin resistance. The presence of the yhcS::TN<Kan>mutation in one of the resultant transductants, named DPD2433, wasconfirmed by PCR amplification. Plasmid pDEW655 was moved to E. colistrains MG1655 and DPD2433 by transformation, selecting for Ampicillinresistance to generate strains DPD2436 and DPD2437, respectively. Thebioluminescent response of these two strains to pHBA was tested.Aliquots (50 μl) of actively growing cultures at 37° C. in LB mediumthat had been previously diluted, and from overnight cultures in LBmedium with 150 μg/ml Ampicillin were added to 50 μl of LB medium at pH7.0 containing PHBA as the sodium salt form. Several concentrations ofpHBA were tested. Table 4 below shows the response in these two hoststrains at thirty minutes after cells were added to pHBA containingmedium. TABLE 4 Bioluminescence response of the yhcRQP-luxCDABE genefusion [pHBA], RLU Ratio treated/control mM yhcS+ yhcS− yhcS+ yhcS− 1000.437 0.045 0.693 0.055 50 91.7 0.614 145 0.753 25 66.6 1.59 106 1.9512.5 30.8 1.82 48.8 2.23 6.2 16.2 1.42 25.7 1.75 3.1 10.2 1.16 16.2 1.421.6 6.72 1.02 10.6 1.25 0 0.631 0.815 1 1

[0137] The yhcS::TN<Kan> mutation almost completely eliminated theupregulation of expression induced by pHBA treatment at allconcentrations tested. Also note that in the yhcS⁺ strain, the level ofgene expression as quantitated by the degree of bioluminescence variedwith the concentration of PHBA added. Thus, the amount of inducer addedcan be used to tune the expression level from this promoter.

[0138] Overall, in two different E. coli host strains, a functional YhcSwas required for upregulation of yhcRQP expression in response to pHBAaddition. These results prove that YhcS is a positive acting factor forupregulation of transcription of the yhcRQP operon.

Example 4 Structure activity relationships for YhcS activation

[0139] Further characterization of the signals that trigger activationof YhcS, was done using the yhcRQP-luxCDABE gene fusion containing E.coli strains DPD241 1 or DPD2436. Table 5 shows the results ofbioluminescence activation tests done with cells in LB medium at pH 7.0,as described in Example 3. Several weak, aromatic acid molecules inaddition to those shown in the Examples above activated expression.Thus, the known inducing molecules comprise PHBA, pHCA, CA, salicylate,benzoate, and 1-napthoate. TABLE 5 Upregulation of yhcRQP-luxCDABEexpression by aromatic carboxylic acids Ex- Concen- peri- tration mentE. coli of maximum treated control code* strain Compound response RLURLU Ratio A DPD2411 1- 2.5 mM  29.662 0.2244 132 napthoate A DPD2411Sodium 25 mM 69.678 0.231 302 pHBA B DPD2436 Sodium 6.2 mM  3.759 0.289813 benzoate C DPD2436 Sodium 12.5 mM   8.093 0.6792 12 benzoate CDPD2436 Sodium 50 mM 91.658 0.6311 145 pHBA D DPD2436 Sodium 6.2 mM 14.703 0.1908 77 salicylate D DPD2436 Sodium 50 mM 48.643 0.196 248 pHBA

[0140] Compounds tested that did not induce expression were defined asthose for which there resulted less than 3-fold increase in lightproduction from E. coli strains containing pDEW655. Compounds unrelatedin structure to the known inducing molecules did not induce expression.Those tested were acetate, propionate, ethanol, limonene, NaCl,polymyxin sulfate, benzalkonium chloride, gramicidin S, and SDS. Inaddition, several compounds related in structure to the inducingmolecules were not inducers, including methyl paraben,p-hydroxystryrene, 2-biphenylcarboxylate, and L-tyrosine. Thus, therequirement for the carboxylate moiety was demonstrated by the lack ofresponse to methyl paraben, the methyl ester of PHBA, and top-hydroxystryrene, a molecule related to pHCA but lacking thecarboxylate group. The requirement for an aromatic ring was demonstratedby the lack of response to non-aromatic carboxylic acids, acetate andpropionate.

[0141] The response of this regulatory system is specific for certainaromatic carboxylic acids. This class of molecules includes compoundsthat are environmentally friendly and relatively inexpensive, such assodium benzoate.

Example 5 Internal acidification is not the signal that activates YhcS

[0142] All characterized activators of YhcS are weak acids such as PHBAand PHCA. Thus, the inducing condition could potentially be eitheracidification of the cytoplasm or presence of the conjugate molecule.The fact that non-aromatic weak acids propionate and acetate, which areknown to cause cytoplasmic acidification, did not induce expression ofthe yhcRQP-luxCDABE gene fusion suggested that cytoplasmic acidificationwas not the inducing signal. This conclusion was confirmed byexperiments comparing upregulation of yhcRQP-luxCDABE mediated by YhcSto that mediated by other well-known acidification responsive regulatorycircuits. Three E. coli strains, each in the same host strain butcarrying different, plasmid-borne, promoter-luxCDABE fusions were used.Strain DPD2411 contains a yhcRQP-luxCDABE gene fusion as described inExample 2. Strain DPD2084 contains a yciG-luxCDABE gene fusion that hasbeen previously described. Strain DPD3282 contains a lysU-luxCDABE genefusion that was part of the LuxA collection of gene fusions, describedin Example 2. The plasmid in this strain, pDEW558, contains an E. colichromosomal segment between nucleotides 4350990 and 4353107 according tothe E. coli genomic sequence; the orientation of the chromosomal segmentis such that the lysU promoter region controls expression of luxCDABE.Each of these three strains was grown overnight in Vogel-Bonner minimalmedium with 0.4% glucose as the carbon source and supplemented withL-proline, L-lysine, uracil and 25 μg/ml Ampicillin. The overnightcultures were diluted into the same medium except lacking Ampicillin andincubated at 37° C. until in mid-exponential growth. Aliquots (50 μl) ofthese actively growing cultures were added to 50 μl of the same mediumat pH 7.0 without Ampicillin but containing various concentrations ofsodium acetate or sodium salicylate in the wells of a 96 well, whitemicroplate. (Microlite, Dynex Technologies). Immediately after addingthe cell culture, the bioluminescence was quantitated in a microplateluminometer in the kinetic mode. Table 6 shows the results at 100minutes after acetate addition or salicylate addition. Treatment of E.coli with acetate did not activate expression of yhcRQP-luxCDABE, butdid activate the other two acid responsive regulatory circuits.Conversely, addition of sodium salicylate upregulated expression ofyhcRQP-luxCDABE, but did not increase expression of the other two acidresponsive gene fusions at the concentrations tested. Thus, it can beconcluded that YhcS is not responding to acidification signals, butrather is responding to the presence of the aromatic molecules. TABLE 6Comparison of acid inducible gene fusions and yhcRQP- luxCDABE responsesto acetate and salicylate RLU at 100 minutes 0 mM 0.6 mM 5.0 mM Gene 0mM 80 mM 160 mM salic- salic- salic- fusion acetate acetate acetateylate ylate ylate yhcRQP- 0.13 0.14 0.08 0.32 7.9 26.0 luxCDABE yciG-0.19 0.35 0.80 0.18 0.19 0.13 luxCDABE lysU- 1.3 1.4 6.0 1.3 1.3 0.89luxCDABE

[0143]

1 9 1 930 DNA Escherichia coli CDS (1)..(930) 1 atg gaa cga cta aaa cgcatg tcg gtg ttt gcc aaa gta gtt gaa ttt 48 Met Glu Arg Leu Lys Arg MetSer Val Phe Ala Lys Val Val Glu Phe 1 5 10 15 ggc tct ttt acc gcc gccgcc aga cag cta cag atg agc gtt tcg tcc 96 Gly Ser Phe Thr Ala Ala AlaArg Gln Leu Gln Met Ser Val Ser Ser 20 25 30 atc agt cag acg gta tca aaactg gaa gat gag ttg cag gta aag ctg 144 Ile Ser Gln Thr Val Ser Lys LeuGlu Asp Glu Leu Gln Val Lys Leu 35 40 45 tta aac cgt agc aca cgc agc attggc ctg acc gaa gcc ggt aga att 192 Leu Asn Arg Ser Thr Arg Ser Ile GlyLeu Thr Glu Ala Gly Arg Ile 50 55 60 tac tac cag ggc tgc cgt cgt atg cttcat gaa gtg cag gat gtt cat 240 Tyr Tyr Gln Gly Cys Arg Arg Met Leu HisGlu Val Gln Asp Val His 65 70 75 80 gag caa ctg tat gcc ttc aat aac accccc atc ggg acg cta cgc att 288 Glu Gln Leu Tyr Ala Phe Asn Asn Thr ProIle Gly Thr Leu Arg Ile 85 90 95 ggc tgt tct tca act atg gca caa aat gttctc gcc ggg ctg aca gcc 336 Gly Cys Ser Ser Thr Met Ala Gln Asn Val LeuAla Gly Leu Thr Ala 100 105 110 aaa atg ctg aaa gaa tac cca ggt ttg agcgtc aat ctg gtt acc gga 384 Lys Met Leu Lys Glu Tyr Pro Gly Leu Ser ValAsn Leu Val Thr Gly 115 120 125 att cca gcc ccc gac ctg att gcc gac ggtctg gat gtg gtg atc cgc 432 Ile Pro Ala Pro Asp Leu Ile Ala Asp Gly LeuAsp Val Val Ile Arg 130 135 140 gtc ggc gcg ttg cag gat tcc agc ctg ttttcc cgc cgt ctg ggc gcg 480 Val Gly Ala Leu Gln Asp Ser Ser Leu Phe SerArg Arg Leu Gly Ala 145 150 155 160 atg cca atg gtg gtg tgc gcc gcg aaaagc tat ctc aca caa tac ggc 528 Met Pro Met Val Val Cys Ala Ala Lys SerTyr Leu Thr Gln Tyr Gly 165 170 175 ata ccg gaa aaa ccc gcc gat ttg agtagt cat tca tgg ctt gaa tac 576 Ile Pro Glu Lys Pro Ala Asp Leu Ser SerHis Ser Trp Leu Glu Tyr 180 185 190 agc gtg cgg ccc gac aat gaa ttt gaactg atc gca ccg gaa ggg atc 624 Ser Val Arg Pro Asp Asn Glu Phe Glu LeuIle Ala Pro Glu Gly Ile 195 200 205 tcg act cgc ctg atc cca caa gga agattt gtg act aat gat ccg atg 672 Ser Thr Arg Leu Ile Pro Gln Gly Arg PheVal Thr Asn Asp Pro Met 210 215 220 acg ctg gtg cgc tgg ctg acg gcg ggtgcc ggg atc gcc tac gtg ccg 720 Thr Leu Val Arg Trp Leu Thr Ala Gly AlaGly Ile Ala Tyr Val Pro 225 230 235 240 ctg atg tgg gtg atc aac gag atcaat cgt ggg gag ctg gag atc ctg 768 Leu Met Trp Val Ile Asn Glu Ile AsnArg Gly Glu Leu Glu Ile Leu 245 250 255 ctg ccg cgt tac cag tca gat ccacgc ccg gtt tat gcg tta tat acc 816 Leu Pro Arg Tyr Gln Ser Asp Pro ArgPro Val Tyr Ala Leu Tyr Thr 260 265 270 gaa aaa gat aag ctg ccg ctg aaggta cag gtc gtg atc aac tcg ctg 864 Glu Lys Asp Lys Leu Pro Leu Lys ValGln Val Val Ile Asn Ser Leu 275 280 285 acg gat tat ttt gtt gag gtc ggtaaa ttg ttt cag gag atg cac ggg 912 Thr Asp Tyr Phe Val Glu Val Gly LysLeu Phe Gln Glu Met His Gly 290 295 300 cgc ggg aaa gag aag taa 930 ArgGly Lys Glu Lys 305 2 309 PRT Escherichia coli 2 Met Glu Arg Leu Lys ArgMet Ser Val Phe Ala Lys Val Val Glu Phe 1 5 10 15 Gly Ser Phe Thr AlaAla Ala Arg Gln Leu Gln Met Ser Val Ser Ser 20 25 30 Ile Ser Gln Thr ValSer Lys Leu Glu Asp Glu Leu Gln Val Lys Leu 35 40 45 Leu Asn Arg Ser ThrArg Ser Ile Gly Leu Thr Glu Ala Gly Arg Ile 50 55 60 Tyr Tyr Gln Gly CysArg Arg Met Leu His Glu Val Gln Asp Val His 65 70 75 80 Glu Gln Leu TyrAla Phe Asn Asn Thr Pro Ile Gly Thr Leu Arg Ile 85 90 95 Gly Cys Ser SerThr Met Ala Gln Asn Val Leu Ala Gly Leu Thr Ala 100 105 110 Lys Met LeuLys Glu Tyr Pro Gly Leu Ser Val Asn Leu Val Thr Gly 115 120 125 Ile ProAla Pro Asp Leu Ile Ala Asp Gly Leu Asp Val Val Ile Arg 130 135 140 ValGly Ala Leu Gln Asp Ser Ser Leu Phe Ser Arg Arg Leu Gly Ala 145 150 155160 Met Pro Met Val Val Cys Ala Ala Lys Ser Tyr Leu Thr Gln Tyr Gly 165170 175 Ile Pro Glu Lys Pro Ala Asp Leu Ser Ser His Ser Trp Leu Glu Tyr180 185 190 Ser Val Arg Pro Asp Asn Glu Phe Glu Leu Ile Ala Pro Glu GlyIle 195 200 205 Ser Thr Arg Leu Ile Pro Gln Gly Arg Phe Val Thr Asn AspPro Met 210 215 220 Thr Leu Val Arg Trp Leu Thr Ala Gly Ala Gly Ile AlaTyr Val Pro 225 230 235 240 Leu Met Trp Val Ile Asn Glu Ile Asn Arg GlyGlu Leu Glu Ile Leu 245 250 255 Leu Pro Arg Tyr Gln Ser Asp Pro Arg ProVal Tyr Ala Leu Tyr Thr 260 265 270 Glu Lys Asp Lys Leu Pro Leu Lys ValGln Val Val Ile Asn Ser Leu 275 280 285 Thr Asp Tyr Phe Val Glu Val GlyLys Leu Phe Gln Glu Met His Gly 290 295 300 Arg Gly Lys Glu Lys 305 3393 DNA Escherichia coli 3 ttcaacctca aacgaacagt cgcgatatca aataaaacaagcagcaatag agcgcggtgt 60 tgaacaacgc cggatgccag acaaagtcgt agatacctgttggcacaagt acccggcgca 120 ccagccagaa aatcgccagt gataaaagca attcaaaaaatatcggtggg aaggacagcc 180 caaacaccac gataacggga aacagactca tgttgaccttggttgtaaag agagagcagg 240 cgttattatt ttcagcatct gtcgccgcag agaagggcatggaaagccgg gcgagagcaa 300 cattgctgta gattgatatt taatatatta gcgtaactgttatgctgtta tctatattat 360 gtgatctaaa tcacttttaa gtcagagtga ata 393 4 24DNA Artificial Sequence Primer 4 gacggcggct ttgttgaata aatc 24 5 25 DNAArtificial Sequence Primer 5 ttggaattta atcgcggcct cgagc 25 6 25 DNAArtificial Sequence Primer 6 acctacaaca aagctctcat caacc 25 7 25 DNAArtificial Sequence Primer 7 gcaatgtaac atcagagatt ttgag 25 8 20 DNAArtificial Sequence Primer 8 aacgcatgtc ggtgtttgcc 20 9 25 DNAArtificial Sequence Primer 9 cgtgcatctc ctgaacaatt taccg 25

We claim:
 1. A method for the inducible expression of a heterologousnucleic acid molecule comprising: a) providing a host cell having agenome comprising: i) a yhcS regulator gene responsive to an aromaticcarboxylic acid inducer; ii) a promoter region, responsive to expressionof the yhcS regulator gene; and iii) at least one heterologous nucleicacid molecule; wherein the at least one heterologous nucleic acidmolecule is operably linked to the promoter region; b) contacting thehost cell of (a) with an aromatic carboxylic acid inducer wherein the atleast one heterologus nucleic acid molecule is expressed.
 2. A methodaccording to claim 1 wherein the at least one heterologous nucleic acidmolecule encodes at least one protein.
 3. A method according to claim 2wherein the at least one protein is part of an enzymatic biosyntheticpathway producing a product selected from the group consisting ofisoprenoids, terpenoids, tetrapyrroles, polyketides, vitamins, aminoacids, fafty acids, proteins, nucleic acids, carbohydrates,antimicrobial agents, and anticancer agents.
 4. A method according toclaim 1 wherein the at least one heterologous nucleic acid moleculeencodes a reporter.
 5. A method according to claim 4 wherein thereporter is selected from the group consisting of luxCDABE, bgaB, cat,dsRed, galk, gfp, lacZ, luc, luxAB, nptil, phoA, uidA, and xylE.
 6. Amethod according to claim 1 wherein the aromatic carboxylic acid induceris selected from the group consisting of para-hydroxybenzoic acid,para-hydroxycinnamic acid, cinnamic acid, salicylic acid, benzoic acid,and 1-napthoic acid.
 7. A method according to claim 1 wherein thepromoter region, responsive to expression of the yhcS regulator gene ispromoter region isolated from the yhcRQR operon.
 8. A method accordingto claim 1 wherein the host cell is an enteric bacteria.
 9. A methodaccording to claim 1 wherein the host cell is selected from the group ofgenera consisting of Escherichia, Salmonella, Bacillus, Acinetobacter,Streptomyces, Methylobacter, Rhodococcus, Corynebacterium, Pseudomonas,Rhodobacter, and Synechocystis.
 10. A method for the inducibleexpression of a heterologous nucleic acid molecule comprising: a)providing an enteric bacterial host cell having a genome comprising: i)a yhcS regulator gene responsive to an aromatic carboxylic acid inducer;ii) a promoter region, responsive to expression of the yhcS regulatorgene; and iii) at least one heterologous nucleic acid molecule; whereinthe at least one heterologous nucleic acid molecule is operably linkedto the promoter region; b) contacting the host cell of (a) with anaromatic carboxylic acid inducer wherein the at least one heterologusnucleic acid molecule is expressed.
 11. A method according to claim 10wherein the yhcS regulator gene responsive to an aromatic carboxylicacid inducer is an isolated nucleic acid molecule selected from thegroup consisting of: a) an isolated nucleic acid molecule comprisingnucleic acid sequence SEQ ID NO:1; and b) an isolated nucleic acidmolecule, which hybridizes to SEQ ID NO:1 after being washed with 0.1×SSC, 0.1% SDS at 65° C. and washed with 2×SSC, 0.1% SDS followed by asecond wash in 0.2×SSC, 0.1% SDS.
 12. A method according to claim 10wherein the promoter region, responsive to expression of the yhcSregulator gene is an isolated nucleic acid molecule selected from thegroup consisting of: a) an isolated nucleic acid molecule comprisingnucleic acid sequence SEQ ID NO:3; and b) an isolated nucleic acidmolecule, which hybridizes to SEQ ID NO:3 after being washed with 0.1×SSC, 0.1% SDS at 65° C. and washed with 2×SSC, 0.1% SDS followed by asecond wash in 0.2×SSC, 0.1% SDS.
 13. A method according to claim 10wherein the enteric bacterial host cell is E. coli.
 14. A host cellcomprising: a) a yhcS regulator gene responsive to an aromaticcarboxylic acid inducer having a nucleic acid sequence selected from thegroup consisting of: i) an isolated nucleic acid molecule comprisingnucleic acid sequence SEQ ID NO:l; and ii) an isolated nucleic acidmolecule, which hybridizes to SEQ ID NO:1 after being washed with 0.1×SSC, 0.1% SDS at 65° C and washed with 2×SSC, 0.1% SDS followed by asecond wash in 0.2×SSC, 0.1% SDS; b) a promoter region, responsive toexpression of the yhcs regulator gene having a nucleic acid sequenceselected from the group consisting of: i) an isolated nucleic acidmolecule comprising nucleic acid sequence SEQ ID NO:3; and ii) anisolated nucleic acid molecule, which hybridizes to SEQ ID NO:3 afterbeing washed with 0.1 ×SSC, 0.1% SDS at 65° C. and washed with 2×SSC,0.1% SDS followed by a second wash in 0.2×SSC, 0.1% SDS; and iii) atleast one heterologous nucleic acid molecule; wherein the at least oneheterologous nucleic acid molecule is operably linked to the promoterregion.
 15. The host cell of claim 14 wherein the host cell is anenteric bacteria.
 16. The host cell of claim 14 wherein the at least oneheterologous nucleic acid molecule encodes at least one protein.
 17. Thehost cell of claim 14 wherein the at least one heterologous nucleic acidencodes a reporter.
 18. The host cell of claim 14 wherein the reporteris selected from the group consisting of luxCDABE, bgaB, cat, dsRed,galK, gfp, lacZ, luc, luxAB, nptll, phoA, uidA, and xylE.
 19. The hostcell of claim 16 wherein the at least one protein is part of anenzymatic biosynthetic pathway producing a product selected from thegroup consisting of isoprenoids, terpenoids, tetrapyrroles, polyketides,vitamins, amino acids, fatty acids, proteins, nucleic acids,carbohydrates, antimicrobial agents, and anticancer agents.